The long term objective of this project is to understand the role of ubiquilin in the pathogenesis of late-onset Alzheimer's disease (AD). AD, the most common age-related dementia, is associated with extracellular deposition and aggregation of proteolytic fragments of amyloid precursor protein (APP). It is believed that these changes are associated with progressive neuronal dysfunction, dementia, and death. Therefore, understanding the molecular events directing the proteolytic processing of APP is essential to understanding the pathogenesis of this disease. Although the mechanism(s) of altered APP processing is (are) beginning to be understood in cases of early-onset familial AD, an overwhelming majority of AD cases are sporadic, or late-onset, with unknown etiology. Recent studies have reported a genetic link between late-onset AD and polymorphisms in the gene encoding ubiquilin. Ubiquilin contains a ubiquitin-like domain, which directly interacts with the proteasome, and it has been implicated in the regulation of the degradation of several proteins. Preliminary studies indicate that ubiquilin binds APP in a yeast-two-hybrid screen. The central hypothesis of this proposal is that ubiquilin contributes to AD by interacting with APP and altering the processing and/or turnover of its potentially toxic cleavage products. The specific aims of this project are as follows: 1) Determine the requirements for ubiquilin to bind APP. Deletion and point-mutation constructs will be generated, and subcellular fractionation and co-immunoprecipitation experiments will be performed to determine the residues necessary for binding and the localization of the interaction. 2) Test whether ubiquilin affects APP processing and/or turnover. Western blotting and ELISA will be used to examine proteolytic fragment production in the presence and absence of ubiquilin. APP turnover will be examined using cycloheximide chase experiments in cultured cell lines and immunocytochemistry/fluorescence tracking in primary neurons. The role of the proteasome will be investigated using proteasome inhibitors. 3) Determine whether ubiquilin can protect cells from APP-induced cytotoxicity. APP-induced cell death in cultured cell lines and primary neurons will be determined in the presence and absence of ubiquilin. Apoptosis assays and fluorescence microscopy will be used. The role of the proteasome will be investigated using proteasome inhibitors. The purpose of this proposal is to examine a novel regulatory role for ubiquilin in the processing of APP. The results of these studies will contribute to our understanding of the pathogenesis of AD and may lead to the development of new therapeutics.